Glaucoma represents the second leading cause of blindness worldwide. While both age and intraocular pressure (IOP) are well-recognized risk factors for this disease, the underlying pathologic process involves accelerated death of retinal ganglion cells (RGC) that is associated with progressive loss of vision. For several decades loss of RGCs has been explained primarily by injury to axons in the optic nerve head (ONH) due to the anatomic and mechanical features of the lamina cribrosa, the specialized ONH zone comprised of collagen beams that define the channels or pores through which axon bundles exit the eye. Many studies have examined the effects of IOP on the lamina cribrosa; however, the histologic techniques used involving fixation, embedding and serial sectioning have precluded direct study of IOP effects on collagen beam organization and channel/pore size. The development of confocal microscopy, which optically sections tissue, has provided a new, non-invasive method for dynamically evaluating the 3-dimensional organization of tissue using digital imaging and reconstruction algorithms. Importantly, recent advances in multiphoton confocal microscopy using femtosecond lasers with high-energy pulses that generate second harmonic (SH) signals from collagen allows for direct optical imaging of the lamina cribrosa, non-invasively over time. The application of SH imaging microscopy (SHIM) to the study of the ONH provides for the first time the ability to test the general hypothesis that increasing intraocular pressure in the same eye results in the independent movement of ONH collagen beams leading to distortion of the lamina cribrosa channels and compression of the axon bundles. To test this hypothesis we propose to: 1. Develop an experimental chamber that will allow pressurizing fresh, unfixed human ONH and allow its dynamic examination by SHIM. 2. Determine the effects of changing pressure on lamina cribrosa pore size and shape in individual human ONH as a function of depth and location. We believe that demonstrated success in our ability to visualize the lamina cribrosa using SHIM will strongly support expanded study of the mechanical properties of lamina cribrosa and the consequent effects of age and ethnicity, important predictors of progressive glaucoma damage. Glaucoma causes progressive loss of vision accompanied by changes in the structure of the optic nerve head typically described as increased cupping. Advancing age and increasing eye pressure are risk factors for progression of glaucoma. We propose to use a new technology to visualize the optic nerve head with high resolution and non-invasively. This technique allows for direct measurement of the structural changes in the associated with glaucoma and has direct clinical relevance and application. [unreadable] [unreadable] [unreadable]